Initiation of DNA Replication in Mammalian Cells

DNA复制小组旨在了解细胞周期机制的信息如何导致DNA复制的开始。正常的细胞生长依赖于相互作用的分子网络，在不利条件下阻止DNA复制和细胞分裂。该网络各组成部分之间错综复杂的平衡被破坏可能导致癌症；然而，干扰细胞周期信号网络传递的信号是癌症治疗的重要工具。更好地了解细胞周期对于制定合理的、以知识为基础的战略来对抗癌症和利用干细胞改善人类健康至关重要。为了在染色质水平上研究细胞周期信号，我们指定了DNA序列，这些序列决定了复制是否、在哪里以及何时发生。决定复制起始位置的DNA序列被称为复制子。当复制子从其原始基因组位点转移到异位基因组位点时，它们开始复制的能力被识别[Aladjem， MI，等]。科学[j].北京：北京大学，1998。复制子的遗传解剖（见特异性目标1）描述了开始DNA复制的序列要求。今年，我们开始详细分析这些序列的dna -蛋白质相互作用。我们还报道（Specific Aim 2），细胞周期s期DNA复制的时间可以被改变[Lin CM， et al.]。[j].中国生物医学工程学报，2009,31(2):393 - 398。我们现在改变复制时间作为一种工具来阐明决定复制时间的遗传和表观遗传因素。我们最近开始使用DNA复制的单分子分析来确定特定序列的复制时间（Specific Aim 2），并评估代谢条件变化和暴露于抗癌药物对起始模式的影响[Shimura T， et al.]。中国生物医学工程学报（英文版）；2009；[375:1152-64m 2008]（具体目标3）。上述研究提供了细胞周期机制与染色质的相互作用，以控制正常生长过程中的DNA复制和对复制干扰药物的反应。以下是最近研究结果的摘要，对每个具体目标进行了简要总结。1)复制因子的特征，影响复制起始位置的遗传因子。我们已经确定，人类β -珠蛋白位点内的复制起始区域包含两个独立的、不重叠的复制子，并且已经确定了这些复制子内DNA复制起始所需的序列基序[Wang L， et al.]。中国生物医学工程学报，2009,33(3):387 - 398。在β -珠蛋白位点，这些序列基序相互作用以确定复制起始事件的位置，这意味着哺乳动物复制因子具有模块化结构[Wang L， et al.]。[j].中国生物医学工程学报，2006,31(2):393 - 398。上述研究与后生动物的复制模式是由序列修饰和表观遗传修饰的组合动态调节的新认识相一致[Aladjem MI.生物学报，8:588-600,2007]。我们最近的观察表明，复制因子在细胞周期的特定阶段结合特定的蛋白质。我们已经确定了一种蛋白质复合物，它通过结合复制子中的特定序列来影响复制时间和基因沉默。我们有证据表明，第二个复制子结合复合体直接影响复制起始事件的位置。我们目前正在参与鉴定第二种蛋白质复合物，并研究这两种蛋白质-DNA相互作用在DNA复制早期阶段的作用。2) DNA序列和染色质结构对复制时间的影响分析。我们已经确定了影响DNA复制时间的DNA序列[Lin CM， et al.]。中国生物医学工程学报（英文版）；2003；冯玉青，等。冯玉清，等。研究表明，DNA复制的时间与染色质凝聚状态和表观遗传因素（如CpG序列的甲基化）有关[j]。林志明，等。组蛋白修饰的研究进展[j]。中国生物医学工程学报（英文版）；2003；付海，等。生物工程学报，2006,24(2):572- 576。即使在不保存复制起始模式的位点上，组织特异性复制时间模式也可以在进化中保守[Aladjem MI， et al.]。中国生物医学工程学报（英文版），2009,31(2):444 - 444。我们已经证明，功能性复制子序列（而不是突变复制子）可以阻止基因沉默和复制延迟，并禁止染色质凝聚[Fu H，等]。生物工程学报，2006,24(2):572- 576。我们发现蛋白质-DNA相互作用会影响DNA沉默，并对参与这种相互作用的蛋白质进行了表征。我们已经证明，阻止这种相互作用的点突变会影响DNA复制的时间，并允许基因沉默。此外，我们所描述的蛋白质-DNA相互作用在确定DNA甲基化如何影响转录沉默方面发挥作用。这些研究正在审查中，准备发表。此外，我们继续与基因治疗实验室合作，在基因治疗载体的开发中评估复制因子对基因沉默的影响的相关性。3) DNA复制扰动诱导的细胞信号相互作用的鉴定。我们发现了一个涉及BLM解旋酶、Mus81核酸酶、ATR激酶和非同源重组级联的细胞反应途径。该途径对暴露于轻度药物诱导的DNA复制扰动（低于细胞周期检查点反应的阈值）后的DNA复制扰动作出反应。暴露于DNA复制轻微扰动的细胞表现出由Mus81在BLM解旋酶和ATR激酶的合作下形成的短暂DNA断裂[Shimura T， et al]。中国生物医学工程学报（英文版），2009:344 - 344。在包含完整的非同源末端连接途径的细胞中，这些DNA断裂是短暂的，细胞在抑制剂的存在下迅速恢复复制，尽管速度很慢。DNA断裂在缺乏非同源末端连接途径组分（如DNA- pk和XRCC4）的细胞中持续存在；在DNA合成受到轻度抑制后，这些细胞无法恢复DNA复制并激活细胞周期检查点反应[Shimura T， et al.]。中国生物医学工程杂志[J]。这些最近的发现表明，复制诱导的DNA断裂并不总是被动地产生于聚合酶碰撞；断裂也可以在干扰复制的分解过程中作为中间体形成。复制起始事件的大规模平行测序研究也表明，不能产生和处理断裂的细胞在正常生长过程中具有独特的DNA复制谱。

The DNA Replication Group aims to understand how information from the cell cycle machinery leads to the initiation of DNA replication. Proper cell growth depends on a network of interacting molecules that prevents DNA replication and cell division under unfavorable conditions. Disruptions in the intricate balance between components of this network may lead to cancer; however, interfering with signals transmitted by the cell cycle signaling network is an important tool for cancer therapy. A better understanding of the cell cycle is fundamental to the development of rational, knowledge-based strategies to combat cancer and utilize stem cells to improve human health. To study cell cycle signaling at the chromatin level, we specify DNA sequences that determine whether, where, and when replication will occur. DNA sequences that determine the location of replication initiation are called replicators. Replicators are identified by their ability to start replication when transferred from their original genomic locus to ectopic genomic sites [Aladjem, MI, et al. Science 281: 1005-9, 1998]. Genetic dissection of replicators (see Specific Aim 1) delineates the sequence requirements for starting DNA replication. This year we have started a detailed analysis of DNA-protein interactions of these sequences. We also report (Specific Aim 2) that the timing of DNA replication during the S-phase of the cell cycle can be altered [Lin CM, et al. Curr Biol 13: 1019-28, 2003]. We now alter replication timing as a tool to elucidate genetic and epigenetic factors that determine replication timing. We have recently started to use single molecule analyses of DNA replication to determine replication timing of particular sequences (Specific Aim 2) and to evaluate the effect of changes in metabolic conditions and exposure to anti-cancer drugs on initiation patterns [Shimura T, et al. J Mol Biol 367: 665-80, 2007; 375:1152-64m 2008] (Specific Aim 3). The studies outlined above provide insights into the interactions of the cell cycle machinery with chromatin to control DNA replication during normal growth and in response to replication-perturbing drugs. Below is a summary of recent findings, summarized briefly for each specific aim. 1) Characterization of replicators, genetic elements that affect the location of replication initiation. We have established that the replication initiation region within the human beta-globin locus contains two independent, non-overlapping replicators and have identified sequence motifs that are required for initiation of DNA replication within these replicators [Wang L, et al. Mol Cell Biol 24, 3373-86, 2004]. At the beta-globin locus, those sequence motifs interact with each other to determine the location of replication initiation events, implying a modular structure for mammalian replicators [Wang L, et al. Hum Mol Genet 15: 2613-22, 2006]. The above studies are in line with the emerging understanding that replication patterns in metazoans are dynamically regulated by a combination of sequence and epigenetic modifications [Aladjem MI. Nat Rev Genet 8: 588-600, 2007]. Our recent observations suggest that replicators bind particular proteins at specific stages of the cell cycle. We have identified a protein complex that affects replication timing and gene silencing by binding to a specific sequence in a replicator. We have evidence suggesting that a second replicator binding complex directly affect the location of replication initiation events. We are currently involved in identification of the second protein complex and in studying the role of the two protein-DNA interactions in the early stages of DNA replication. 2) Analysis of the effect of DNA sequence and chromatin structure on replication timing. We have identified DNA sequences that affect the timing of DNA replication [Lin CM, et al. Curr Biol 13: 1019-28, 2003; Feng YQ, et al. Mol Cell Biol 25: 3864-74, 2007] and have shown that the timing of DNA replication correlates with the status of chromatin condensation and with epigenetic factors, such as methylation of CpG sequences [Feng YQ, et al. PLoS Genet 2, e65, 2006] and histone modifications [Lin CM, et al. Curr Biol 13: 1019-28, 2003; Fu H, et al. Nat Biotechnol 24: 572-6, 2006]. Tissue-specific patterns of replication timing can be conserved in evolution even in loci that do not conserve replication initiation patterns [Aladjem MI, et al. Mol Cell Biol 22: 442-5, 2002]. We have shown that functional replicator sequences (but not mutated replicators) prevented gene silencing and replication delay and prohibited chromatin condensation [Fu H, et al. Nat Biotechnol 24: 572-6, 2006]. We found that a protein-DNA interaction affects DNA silencing and characterized the protein involved in that interaction. We hve shown that point mutations that prevent this interaction affect the timing of DNA replication and allow gene silencing. In addition, the protein-DNA interaction we have characterized plays a role in determining how DNA methylation affects transcriptional silencing. These studies are under review for publication. In addition, we continue to collaborate with gene therapy laboratories to evaluate the relevance of the effects of replicators on gene silencing in the development of gene therapy vectors. 3) Identification of cellular signaling interactions induced by the perturbation of DNA replication. We have uncovered a cellular response pathway involving BLM helicase, Mus81 nuclease, ATR kinase and the non-homologous recombination cascade. This pathway responds to perturbation of DNA replication following exposure to mild drug-induced perturbation of DNA replication, which is below the threshold of the cell cycle checkpoint response. Cells that are exposed to mild perturbation of DNA replication exhibit transient DNA breaks that are formed by Mus81 with the cooperation of BLM helicase and ATR kinase [Shimura T, et al. J Mol Biol 375: 1152-64, 2008]. In cells that contain an intact nonhomologous end-joining pathway, these DNA breaks are transient and cells rapidly resume replication in the presence of the inhibitor, albeit at a slow rate. DNA breaks persist in cells that are deficient in components of the nonhomologous end-joining pathway such as DNA-PK and XRCC4; such cells are unable to resume DNA replication and activate a cell cycle checkpoint response after a mild inhibition of DNA synthesis [Shimura T, et al. J Mol Biol 367: 665-80, 2007]. These recent findings propose that replication-induced DNA breaks do not always arise passively from polymerase collisions; breaks can also form as intermediates in the resolution of perturbed replication. Massively parallel sequencing studies of replication initiation events also suggest that cells that cannot create and process the breaks have a distinct DNA replication profile during normal growth.